C-type cytochromes (cytochromes c) are the major components of electron transport networks used by both prokaryotes and eukaryotes for respiration. All c-type cytochromes contain CXXCH heme-binding motif (1), and some family members have as many as 27 CXXCH motifs (2). Because specific maturation proteins are required for the functional incorporation of hemes into expressed proteins, it has been difficult to overexpress holo-c-type cytochromes for structural measurements, especially those with multiple heme moieties. Several systems have been previously developed to express c-type cytochromes. Using Escherichia coli, yeast heme lyase was co-expressed with mono-heme cytochromes to obtain their expression with hemes (3,4). Likewise, co-expression of cytochromes c maturation proteins of E. coli permitted the expression of holo-c-type cytochromes with multi-heme in E. coli, albeit at low levels (5). Alternatively, the holo-c-type cytochromes were expressed in Shewanella oneidensis MR-1, taking advantage of the host cell's own mechanism for heme insertion (6,7). In this latter case, much higher levels of expression were obtained for multi-heme c-type cytochromes (6). However, this method uses a ligase-based method for gene cloning, making implementation of this methodology for high-throughput cloning and expression problematic. Further, expression is not regulated, and expressed proteins contain no affinity tag, making subsequent protein purification difficult. We have, therefore, extended this methodology to develop a system suitable for high level expression and purification of the holo-c-type cytochromes with multi-heme.

We used the pBAD202/D-TOPO expression vector from Invitrogen (Carlsbad, CA, USA). This vector is a pUC-like plasmid that replicates in S. oneidensis MR-1 cells. Instead of using the previously described ligase-based method, pBAD202/D-TOPO uses a highly efficient topoisomerase-based strategy for directional cloning of PCR products (8). The expression of the genes cloned into pBAD202/D-TOPO is inducible by L-arabinose (9). pBAD202/D-TOPO also incorporates a V5-epitope and a His6 tag at the C termini of expressed proteins to facilitate subsequent protein detection and isolation.

To determine whether pBAD202/D-TOPO could be used in S. oneidensis MR-1 cells for expression of multi-heme c-type cytochromes, two c-type cytochrome-encoding genes were cloned into pBAD202/D-TOPO according manufacturer's instruction. They were mtrA of S. oneidensis MR-1 and DVU3171 of Desulfovibrio vulgaris Hildenborough ((Table 1)). Since pBAD202/D-TOPO put additional His-patch (HP)-thioredoxin at the N termini of recombinant proteins, a stop codon was added to the forward PCR primer 5′ end immediately after CACC to avoid fusion with HP-thioredoxin. The forward primer also contained a bacterial ribosome binding site ((Table 1)). After they were verified by DNA sequencing, the constructs were used to transform electrocompetent S. oneidensis MR-1 cells at 0.75 kV, 400 ohms, and 25 µF with a Gene Pulser® from Bio-Rad Laboratories (Hercules, CA, USA).

The Shewanella strain with the construct containing mtrA was first used to determine whether expression of recombinant protein was inducible with L-arabinose using enzyme-linked immunofiltration assay (ELIFA) (10). Results of ELIFA showed that expression levels of MtrA increased from less than 0.01% to 0.9% of total soluble proteins 14 h after induction with 1 mM L-arabinose, demonstrating that expression of recombinant protein could be regulated in Shewanella cells. Recombinant MtrA and DVU3171 were purified with Ni2 + -nitrilotriacetic acid (Ni2 + -NTA) agarose (Qiagen, Valencia, CA, USA) to electrophretic homogeneity. Results of Western blot analysis and heme-staining (11) confirmed the identity of heme-containing recombinant MtrA and DVU3171 ((Figure 1), inset; see legend for detail). The absorbance spectra of isolated MtrA and DVU3171 were typical for c-type cytochromes. Oxidized MtrA (data not shown) and DVU3171 had a maximal Soret (γ) absorption peak at 408 nm ((Figure 1), curve 1). Upon reduction, the Soret band maximum shifts to 419 nm and the characteristic and a Soret peaks become more prominent at 523 and 552 nm, respectively ((Figure 1), curve 2). The average heme content of purified MtrA and DVU3171 was calculated at 10.4 for MtrA and 4.1 for DVU3171, which is consistent with the results of previous publication and the prediction based on the numbers of CXXCH heme-binding sites (5,12). In addition, the construct containing mtrA restored the ability of S. oneidensis MR-1 mtrA mutant to reduce iron oxide and grow under electron acceptor-limited condition, indicating that recombinant MtrA was functional in vivo (data not shown). Taken together, these data clearly demonstrate that pBAD202/D-TOPO can be used for overexpression of holo-c-type cytochromes with multi-heme either homologously or heterologously in S. oneidensis MR-1.

Figure 1.

In summary, we have improved a system for protein overexpression of c-type cytochromes containing multi-heme. This system is more efficient than the previous ones in gene cloning and affinity purification of expressed cytochromes. In addition, protein expression in this improved system can be regulated through addition of L-arabinose, making it useful for high-level expression of multi-heme c-type cytochromes. Successful development of this system will help to determine the molecular structures of multi-heme c-type cytochromes, including those found in the bacteria involved in bioremediation of environmental pollutants such as uranium and chromium.